The overall goal of this application is to define conditions that are conducive to generating transplantable, 3-D human lung. Major obstacles to lung transplantation include the paucity of donor lungs available, chronic rejection the requirement for lifelong immunosuppression and high mortality. A new source of transplantable lung tissue that will not be subject to rejection is needed. Using decellularized lung matrix (DLM) as the natural foundational scaffold, our central hypothesis is that building functional, transplantable lung requires a combination of geometry that allows for appropriate cell localization and biomimetic cues superimposed on cells as they align to drive functional integration and maturation. The PI of this application developed a "decellularized, ventilated lung bioreactor" system that supports the growth of fetal lung cells and is amenable to evaluation of the potential to rebuild the lung. In addition to the use of DLM, the novelty of this proposal entails the use of autologous cells with lung recellularization potential all from the same BM source, i.e. all cells are autologous to each other and have the potential to be used in translational clinical studies. Post natal/adult human iPS cells are cells that have been reprogrammed to have characteristics of embryonic stem cells. We have evidence that DLMs can be seeded with induced pluripotent stem cells (iPS cells) that give rise to alveolar type II-like cells. We wish to evaluate the use of autologous BM-derived mesenchymal stromal cells (MSCs) and iPS-derived endothelial cells (ECs) in completing the recellularization of DLM. A team of investigators representing a convergence of the fields of bioengineering, pulmonary biology, physiology, stem cells, vascular biology and thoracic surgery has been assembled. Our aims encompass the recellularization of DLM in a developmental stage-appropriate manner that results in correct cell patterning with tight junctional barriers, gas exchange and fluid clearance functional properties. We will take a stepwise approach to determine the contribution of matrix geometry, physiological stretch and vascularization on re-epithelialization and function of recellularized constructs using human autologous progenitors and compare to native lung. We reason that by comparing different conditions head-to-head and in combination, several fundamental "rules" for bioengineering an autologous functional lung will be defined. There are 3 Specific Aims in this proposal. In Aim 1 we will determine the contribution of matrix geometry and physiological mechanical stretch on the differentiation of human progenitor cells into pulmonary cells. In Aim 2, we will evaluate if recellularization of DLMs is best achieved with mesenchymal induction using BM-derived MSCs. Aim 3 will determine if prior endothelialization of the DLM vascular network potentiates subsequent epithelial progenitor cell attachment to DLM, differentiation and function. We will use BM-derived endothelial cells (ECs) infused through the vasculature access to re-endothelialize DLM. Since vessels are stabilized by pericytes that are of MSC origin, we will use human BM-derived MSCs to study if they can potentiate the attachment of ECs. (End of Abstract)